Research of the past decade evinces a strong and ongoing interest in devising new and better methods for introducing biological materials, in particular, nucleic acids, such as ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and, more recently, peptide nucleic acid (PNA), into eukaryotic cells. Such methods are prerequisites not only for so-called simple gene transfections in vitro, but also for a true understanding of gene function (which requires, in part, an ability to deliberately modify the genome) directed toward an ultimate goal of augmentation therapy or, more preferably, somatic gene modification or therapy in vivo, to correct the underlying defect in inheritable diseases (IDs). Although, many techniques such as use of DEAE-dextran, electroporation, calcium phosphate, microinjection, and osmotic shock are available for in vitro nucleic transfer, these methods are of limited use for in vivo nucleic acid transfer as they are either toxic to cells or their efficiencies of nucleic acid transfer are low (Felgner et al., Proc. Natl. Acad. Sci., 84, 7413-7417 (1987)).
Consequently, "early" researchers of gene therapy (i.e., those within the last few years) resorted to use of retroviral vectors, which are capable of effecting their own means of entry into cells. In the predominant approach to gene modification, researchers almost exclusively delivered nucleic acids ex vivo to isolated cells, and then infused the cells back into the living host. However, retroviruses have a number of drawbacks which severely limit their application, particularly in vivo. For example, since retroviral vectors require target cell proliferation in order to transfer exogenous nucleic acid sequences, they are ineffective for transferring nucleic acids to cells which replicate slowly or are terminally differentiated (Mastrangeli et al., J. Clin. Invest., 91, 225-34 (1993)). Additionally, they integrate randomly into the genome, potentially resulting in loss of control of the subcloned DNA, as well as host genetic alterations due to the disruption of genes. Moreover, retroviruses exhibit restricted host cell range and can be obtained only in relatively low titer (Burns et al., Proc. Natl. Acad. Sci., 90, 8033-37 (1993)).
Consequently, many researchers turned to adenovirus (Ad) to resolve this dilemma, since host cell proliferation is not required for adenoviral gene expression (Horwitz, In: Virology, 2nd Ed., Fields et al., eds., (New York: Raven Press, 1990) 1679-1721; Berkner, K. L., BioTechniques, 6, 606-629 (1988)). Furthermore, Ad possesses tremendous potential for eukaryotic studies given that it can infect a broad range of cell types from a variety of diverse species, it is easy to prepare in high titer, and it can easily be rendered replication-deficient, thus preventing the virus from usurping, and eventually destroying, the target cell (Ginsberg (ed.) The Adenoviruses, (New York: Plenum Press, 1984); Horwitz, In: Virology, 2nd Ed., Fields et al., eds., (New York: Raven Press, 1990) 1679-1721; Rosenfeld et al., Science, 252, 431-434 (1991); Rosenfeld et al., Cell, 68, 143-155 (1992); Quantin et al., Proc. Natl. Acad. Sci., 89, 2581-2584 (1992)).
Additional advantages of adenoviral vectors include that recombination events are rarely observed with use of such vectors. Moreover, despite ubiquitous infection with adenoviruses, an association of such infections with any human malignancy has not been demonstrated. In fact, the live (or non-attenuated) form of adenovirus has been safely employed as a human vaccine ((Horwitz, In: Virology, 2nd Ed., Fields et al., eds., (New York: Raven Press, 1990) 1679-1721; Berkner, K. L., BioTechniques, 6, 606-629 (1988); Ginsberg (ed.) The Adenoviruses, (New York: Plenum Press, 1984)). Furthermore, adenovirus exhibits trophism for the respiratory epithelium, and can be transcribed, translated, and appropriately processed in lung, gastrointestinal (GI), as well as a variety of other types of tissue (Fields et al., eds., (New York: Raven Press, 1990) 1679-1721; Crystal et al., Nucleic Acids Res., 21, 1607-12 (1993)). All these factors suggest that adenovirus constitutes a powerful tool in somatic gene therapy of IDs, particularly those which manifest in disorders of the lung and GI tract.
Human adenovirus exists as a non-enveloped double-stranded DNA virus (Horwitz, In: Virology, 2nd Ed., Fields et al., eds., (N.Y.: Raven Press, 1990) 1679-1721). The adenovirus provides a dramatic example of a naturally evolved and highly efficient mechanism for transferring biological materials to target cells (Otero et al., Virology, 160, 75-80 (1987); FitzGerald et al., Cell, 32, 607-617 (1983); Seth et al., Mol. Cell Biol., 4, 1528-1533 (1984); Yoshimura, Cell Struct. Funct., 10, 391-404 (1985); Defer et al., J. Virol., 64, 3661-3673 (1990); Rosenfeld et al., Science, 252, 431-434 (1991); Curiel et al., Proc. Natl. Acad. Sci., 88, 8850-8854 (1991); Rosenfeld et al., Cell, 68, 143-155 (1992); Quantin et al., Proc. Natl. Acad. Sci., 89, 2581-2584 (1992); Curiel et al., Hum. Gene Therapy, 3, 147-154 (1992)). Namely, Ad enters cells by a receptor-mediated endocytosis (RME) pathway. In the initial virus-receptor interaction in this pathway, Ad binds with specific receptors present on the cell surface via fibers on its outer surface (i.e., the outer shell of each Ad is comprised of 240 hexons and 12 pentons, with each penton being composed of a penton base and a fiber) (Ginsberg (ed.) The Adenoviruses, (New York: Plenum Press, 1984); Horwitz, In: Virology, 2nd Ed., Fields et al., eds., (New York: Raven Press, 1990) 1679-1721; Seth et al., In: Virus Attachment and Entry into Cells, Colwell et al., eds., (WA, D.C.: American Society for Microbiology, 1986) 191-195. Following attachment, the receptors with bound Ad cluster in coated pits, and the virus is internalized within a clathrin-coated vesicle and, subsequently, into an endosomal vesicle, termed an endosome, or receptosome (FitzGerald et al., Cell, 32, 607-617 (1983)).
Within the endosome, the pH of the vesicle is reduced by means of a proton pump associated with the endosomal membrane. The reduced pH effects an alteration in the conformation of the Ad capsid proteins, particularly the penton base protein, which results in disruption of the endosome. As a consequence of this disruption, the endocytic contents, including the Ad, are released into the cytoplasm, and the Ad is then translocated to the nucleus where it directs the synthesis of nascent nucleic acids (FitzGerald et al., Cell, 32, 607-617 (1983); Seth et al., Mol. Cell Biol., 4, 1528-1533 (1984); Seth et al., In: Virus Attachment and Entry into Cells, Colwell et al., eds., (WA, D.C.: American Society for Microbiology, 1986) 191-195; Seth et al., J. Virol., 51, 650-655 (1984); Seth et al., J. Biol. Chem., 259, 14350-14353 (1984); Seth et al., J. Biol. Chem., 260, 9598-9602 (1985); Seth et al., J. Biol. Chem., 260, 14431-14434 (1985); Blumenthal et al., Biochemistry, 25, 2231-2237 (1986); Seth et al., J. Virol., 61, 883-888 (1987)).
This ability of the Ad to easily enter cells has been seized upon as a means of transporting macromolecules into cells (Otero et al., Virology, 160, 75-80 (1987); FitzGerald et al., Cell, 32, 607-617 (1983); Seth et al., Mol. Cell Biol., 4, 1528-1533 (1984); Yoshimura, Cell Struct. Funct., 10, 391-404 (1985); Defer et al., J. Virol., 64, 3661-3673 (1990); Rosenfeld et al., Science, 252, 431-434 (1991); Curiel et al., Proc. Natl. Acad. Sci., 88, 8850-8854 (1991); Rosenfeld et al., Cell, 68, 143-155 (1992); Quantin et al., Proc. Natl. Acad. Sci., 89, 2581-2584 (1992); Curiel et al., Hum. Gene Therapy, 3, 147-154 (1992)). For example, Ads are able to enhance the transfer of a variety of non-viral macromolecules such as dextrans (Otero et al., Virology, 160, 75-80 (1987)), proteins (Carrasco, Virology, 113, 623-629 (1981); Defer et al., J. Virol., 64, 3661-3673 (1990); FitzGerald et al., Cell, 32, 607-617 (1983); Fernandez-Puentes et al., Cell, 20, 769-775 (1980); Otero et al., Virology, 160, 75-80 (1987)), and plasmid DNA linked to ligands (Curiel et al., Hum. Gene Therapy, 3, 147-154 (1992); Cotten et al., Proc. Natl. Acad. Sci., 89, 6094-098 (1992)) to target cells both in vitro and in vivo.
There are two means by which such transfer has been effected. First, the Ad has been employed to transfer non-viral macromolecules packaged within the Ad either in place of, or in addition to, normal Ad components (Berkner, K. L., BioTechniques, 6, 606-629 (1988)). For example, the genome of the Ad has been modified to incorporate exogenous DNA. The recombinant Ad is then packaged to constitute an infectious virus capable of entering cells and transferring the exogenous DNA to the nucleus (Rosenfeld et al., Science, 252, 431-434 (1991); Rosenfeld et al., Cell, 68, 143-155 (1992); Quantin et al., Proc. Natl. Acad. Sci., 89, 2581-2584 (1992); Berkner, K. L., BioTechniques, 6, 606-629 (1988)). Second, the Ad has been employed to mediate the transfer of non-viral macromolecules either linked to the surface of the Ad or, in a "bystander" process where the macromolecule is cointernalized, taken along as cargo in the Ad receptor-endosome complex (Otero et al., Virology, 160, 75-80 (1987); FitzGerald et al., Cell, 32, 607-617 (1983); Seth et al., Mol. Cell Biol., 4, 1528-1533 (1984); Yoshimura, Cell Struct. Funct., 10, 391-404 (1985); Defer et al., J. Virol., 64, 3661-3673 (1990)).
The mechanism by with the Ad augments internalization of non-viral biologic materials is believed to be by increasing the permeability of the target cell plasma membrane (Otero et al., Virology, 160, 75-80 (1987)) or, more likely, by cointernalization of the exogenous biologic material as an "innocent bystander" when the Ad-receptor complexes cluster on the membrane and are internalized (FitzGerald et al., Cell, 32, 607-617 (1983); Seth et al., Mol. Cell Biol., 4, 1528-1533 (1984); Yoshimura, Cell Struct. Funct., 10, 391-404 (1985); Otero et al., Virology, 160, 75-80 (1987); Defer et al., J. Virol., 64, 3661-3673 (1990)). These processes are not Ad-specific, as similar phenomena have been observed with other non-enveloped viruses such as picornavirus (Fernandez-Puentes et al., Cell, 20, 769-775 (1980); Otero et al., Virology, 160, 75-80 (1987); Carrasco, Virology, 113, 623-629 (1981)), as well as enveloped viruses including paramyxovirus, rhabdovirus, poxvirus, and togavirus (Fernandez-Puentes et al., Cell, 20, 769-775 (1980); Otero et al., Virology, 160, 75-80 (1987); Yamaizumi et al., Virology, 95, 216-221 (1979); Carrasco et al., Virology, 117, 62-69 (1982)).
Most of the research attention on virus-mediated cointernalization of macromolecules has been focused on cointernalization of proteins, including toxins and various reporter proteins (Ferna-Puentes et al., Cell, 20, 769-775 (1980); FitzGerald et al., Cell, 32, 607-617 (1983); Seth et al., Mol. Cell Biol., 4, 1528-1533 (1984); Otero et al., Virology, 160, 75-80 (1987); Defer et al., J. Virol., 64, 3661-3673 (1990); Carrasco, Virology, 113, 623-629 (1981); Yamaizumi et al., Virology, 95, 216-221 (1979); Carrasco et al., Virology, 117, 62-69 (1982)). The concept that cointernalization might be employed for Ad-mediated transfer of nucleic acids was suggested, but not evaluated, by Otero and Carrasco (Otero et al., Virology, 160, 75-80 (1987)). In fact, the more recent approaches with respect to transfer of nucleic acids using adenovirus have centered on nucleic acid transfer by attachment of the nucleic acid to molecules capable of effecting its entry into the cell. For instance, in one approach, the nucleic acid is part of a polylysine-glycoprotein carrier complex capable of binding a particular cell surface receptor, or is complexed with a nonspecific ligand such as a charged polypeptide (Rosenfeld et al., Science, 252, 431-434 (1991); Curiel et al., Proc. Natl. Acad. Sci., 88, 8850-8854 (1991); Rosenfeld et al., Cell, 68, 143-155 (1992); Quantin et al., Proc. Natl. Acad. Sci., 89, 2581-2584 (1992); Curiel et al., Hum. Gene Therapy, 3, 147-154 (1992)); Cotten et al., Proc. Natl. Acad. Sci., 89, 6094-098 (1992); Cotten et al., J. Virology, 67, 3777-3785 (1993)). In a more recent approach, the nucleic acid is attached to the outside of the adenoviral capsid by means of conjugation of the nucleic acid through a polylysine residue to the antibody to adenoviral capsid protein (Curiel et al., Human Gene Ther., 3, 147-154 (1992)). Thus, despite this early suggestion by Otero et al., researchers have clearly perceived a lack of feasibility of using adenovirus-driven RME for transfer of nucleic acid. Moreover, the prevailing approaches using adenovirus for transfer of nucleic acids are limited in that the specific receptor to the ligand employed (e.g., transferring must be present on the cell surface for transfection to be accomplished. Additionally, it was discovered recently that better transfection results are obtained when the DNA is not physically attached to any molecule upon introduction into the cell (Wolff et al., Science, 247, 1465 (1990); Acsadi et al., Nature, 352, 815 (1991)). This finding underscores the restrictive nature of current approaches to adenoviral-mediated transfer of DNA to the cell, which require attachment of DNA for cell transfection.
In an essential mimic of the approach of using an adenovirus as a vector, the nucleic acid to be transfected is ensheathed in a virion-like microenvironment. The encased nucleic acid may then be transferred via intracellular injection or, optimally, spontaneous fusion with the cellular membrane. (Nabel et al., Proc. Natl. Acad. Sci., 90, 10759-10763 (1993); Nabel et al., Proc. Natl. Acad. Sci., 90, 11307-11311 (1993)). The microenvironment may be comprised of liposomes or hollow vesicles synthesized using lipids and/or phospholipids (Tikchonenko et al., Gene, 63, 321-330 (1988); Hawley-Nelson et al., Focus, 15, 73-79 (1993); Felgner et al., Proc. Natl. Acad. Sci., 84, 7413-7417 (1987); U.S. Pat. No. 5,264,618). While such an approach is advantageous in that potentially greater amounts of nucleic acids can be transferred, disadvantages of the approach include failure of the liposome to fuse with the cell membrane and degradation of nucleic acids taken up by phagocytosis, and the inherent toxicity of intracellular injection, as well as the toxicity of ether bonds, which may accumulate in the cell as a consequence of liposome-mediated transfection.
Attempts have been made to enhance the ability of the liposomes to fuse with the membrane through subsequent infection with, for example, Sendai virus (Tomita et al., Biochem. Biophys. Res. Comm., 186, 129-34 (1992); Kato et al., J. Biolog. Chem., 266, 3361-364 (1991); Yamaizumi et al., Virology, 95, 218-221 (1979)). However, studies supporting the increased cytotoxicity of exogenously supplied liposomes to cells transformed with adenovirus (Shimura et al., Cancer Research, 48, 578-583 (1988)), suggest against the combination of adenoviral-mediated nucleic acid transfer with liposome-mediated nucleic acid delivery.
There remains a need, therefore, for a method of capitalizing on the inherent ability of adenovirus to effect transport of cargo macromolecules to the cell nucleus by means of RME in a method of transfection that can be employed either in vitro or in vivo, and which avoids the attendant problems of the previously described approaches. It is an object of the present invention to provide such a method of adenoviral-mediated cell transfection with nucleic acids, as well as to provide a means of enhancing this method through use of cationic agents. These and other objects and advantages of the present invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.